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Synthetic cationic lipids have garnered significant attention as promising candidates for gene/DNA transfection in therapeutic applications. The phase behavior of the vesicles formed by these lipids is intriguing, revealing intricate connections to the structure and dynamics of the membrane. These phenomena emerge from the complex interplay between hydrophobic and electrostatic interactions of the lipids. In this study, we explore the impact of an ionic liquid-based surfactant, 1-decyl-3-methylimidazolium bromide (DMIM[Br]), on the structural, dynamical, and phase behavior of cationic dihexadecyldimethylammonium bromide (DHDAB) vesicles. Our investigations indicate that the addition of DMIM[Br] increases the vesicle size while thinning the membrane. Further, DMIM[Br] also induces substantial changes in the membrane phase behavior. At 10 and 25 mol %, DMIM[Br] eliminates the pre-transition from coagel to intermediate crystalline (IC) phase and decreases the onset temperature of the main phase transition to the fluid phase. In the cooling cycle, the addition of DMIM[Br] further induces the formation of an intermediate gel phase. This behavior is reminiscent of the non-synchronous ordering observed in the DODAB membrane, a longer-chain counterpart of DHDAB. Interestingly, at 40 mol % of DMIM[Br], the formation of the intermediate gel phase is largely suppressed. Neutron scattering data provide evidence that the addition of DMIM[Br] enhances lipid mobility in coagel and fluid phases, suggesting that DMIM[Br] acts as a plasticizer, enhancing membrane fluidity across all of the phases. Our findings infer that DMIM[Br] modulates the membrane's phase behavior and fluidity, two essential ingredients for the efficient transport of cargo, by controlling the balance of electrostatic and hydrophobic interactions.
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Ionic liquids based on doubly charged cations, often termed dicationic ionic liquids (DILs), offer robust physicochemical properties and low toxicity than conventional monocationic ionic liquids. In this design-based study, we used solid-state NMR spectroscopy to provide the interaction mechanism of two DILs, 1,n-bis(3-alkylimidazolium-1-yl) alkane dibromide ([C2n(C7-nIM)2]2+·2Br-, n = 1, 6), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipid membranes, to explain the low toxicity of DILs toward HeLa, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cell lines. Dications with a short linker and long terminal chains cause substantial perturbation to the bilayer structure, making them more membrane permeabilizing, as shown by fluorescence-based dye leakage assays. The structural perturbation is even higher than [C12(MIM)]+ monocations, which carry a single 12-carbon long chain and exhibit a much higher membrane affinity, permeability, and cytotoxicity. These structural details are a crucial contribution to the design strategies aimed at harnessing the biological activity of ionic liquids.
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Líquidos Iônicos , Fosfolipídeos , Bicamadas Lipídicas/química , Líquidos Iônicos/toxicidade , Líquidos Iônicos/química , Alcanos/toxicidade , Escherichia coli/metabolismoRESUMO
Curcumin, the main ingredient in turmeric, has attracted attention due to its potential anti-inflammatory, anticancer, wound-healing, and antioxidant properties. Though curcumin efficacy is related to its interaction with biomembranes, there are few reports on the effects of curcumin on the lateral motion of lipids, a fundamental process in the cell membrane. Employing the quasielastic neutron scattering technique, we explore the effects of curcumin on the lateral diffusion of the dipalmotylphosphatidylcholine (DPPC) membrane. Our investigation is also supported by Fourier transform infrared spectroscopy, dynamic light scattering, and calorimetry to understand the interaction between curcumin and the DPPC membrane. It is found that curcumin significantly modulates the packing arrangement and conformations of DPPC lipid, leading to enhanced membrane dynamics. In particular, we find that the presence of curcumin substantially accelerates the DPPC lateral motion in both ordered and fluid phases. The effects are more pronounced in the ordered phase where the lateral diffusion coefficient increases by 23% in comparison to 9% in the fluid phase. Our measurements provide critical insights into molecular mechanisms underlying increased lateral diffusion. In contrast, the localized internal motions of DPPC are barely altered, except for a marginal enhancement observed in the ordered phase. In essence, these findings indicate that curcumin is favorably located at the membrane interface rather than in a transbilayer configuration. Further, the unambiguous evidence that curcumin modulates the membrane dynamics at a molecular level supports a possible action mechanism in which curcumin can act as an allosteric regulator of membrane functionality.
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Curcumina , Bicamadas Lipídicas , 1,2-Dipalmitoilfosfatidilcolina/química , Membrana Celular/química , Curcumina/química , Bicamadas Lipídicas/química , Membranas , Movimento (Física)RESUMO
BACKGROUND: RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. RESULTS: Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. CONCLUSIONS: Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.
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Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , RNA/genética , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , Análise de Sequência de RNARESUMO
Ionic liquids (ILs) are the attractions of researchers today due to their vast area of potential applications. For biomedical uses, it becomes essential to understand their interactions with cellular membrane. Here, the membrane is mimicked with lipid bilayer and monolayer composed of liver lipids extract. Three archetypal imidazolium based ILs, 1-decyl-3-methylimidazolium tetrafluoroborate ([DMIM][BF4] or [C10MIM][BF4]), 1-octyl-3-methylimidazolium tetrafluoroborate, ([OMIM][BF4] or [C8MIM][BF4]) and 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4] or [C2MIM][BF4]) having different alkyl chain lengths are used in the present study. The isothermal titration calorimetry (ITC) measurements showed that [DMIM][BF4] interacts strongest with the liver lipid membrane compared to other two ILs which have relatively shorter alkyl chain length. The low values of stoichiometry ratio of ILs indicates that ILs penetrate within the core of the lipid bilayer. The interaction of ILs with the liver lipid membrane is found to be mainly driven by entropy which could be due to the change in the structure of the lipid membrane at local or global scales. Dynamic light scattering (DLS) measurements indicate that there are no changes in the size of vesicles due to addition of [DMIM][BF4] indicating stability of the vesicles. On the other hand, x-ray reflectivity (XRR) measurements showed a concentration dependent change in the monolayer structure. At low concentration of the IL, the monolayer thickness decreases, exhibiting an increase in the electron density of the layer. However, at higher concentrations, the monolayer thickness increases proving a concentration dependent effects of the IL on the arrangement of the molecules.
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Membrana Celular/química , Líquidos Iônicos/química , Fígado/metabolismo , Animais , Calorimetria , Membrana Celular/metabolismo , Imidazóis/química , Líquidos Iônicos/metabolismo , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
The membrane is one of the key structural materials of biology at the cellular level. Composed predominantly of a bilayer of lipids with embedded and bound proteins, it defines the boundaries of the cell and many organelles essential to life and therefore is involved in almost all biological processes. Membrane-specific interactions, such as drug binding to a membrane receptor or the interactions of an antimicrobial compound with the lipid matrix of a pathogen membrane, are of interest across the scientific disciplines. Herein we present a review, aimed at nonexperts, of the major neutron scattering techniques used in membrane studies: small-angle neutron scattering, neutron membrane diffraction, neutron reflectometry, quasielastic neutron scattering, and neutron spin echo. Neutron scattering techniques are well suited to studying biological membranes. The nondestructive nature of cold neutrons means that samples can be measured for long periods without fear of beam damage from ultraviolet, electron, or X-ray radiation, and neutron beams are highly penetrating, thus offering flexibility in samples and sample environments. Most important is the strong difference in neutron scattering lengths between the two most abundant forms of hydrogen, protium and deuterium. Changing the relative amounts of protium/deuterium in a sample allows the production of a series of neutron scattering data sets, enabling the observation of differing components within complex membrane architectures. This approach can be as simple as using the naturally occurring neutron contrast between different biomolecules to study components in a complex by changing the solution H2O/D2O ratio or as complex as selectively labeling individual components with hydrogen isotopes. This review presents an overview of each experimental technique with the neutron instrument configuration, related sample preparation and sample environment, and data analysis, highlighted by a special emphasis on using prominent neutron contrast to understand structure and dynamics. This review gives researchers a practical introduction to the often enigmatic suite of neutron beamlines, thereby lowering the barrier to taking advantage of these large-facility techniques to achieve new understandings of membranes and their interactions with other molecules.
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Here, we report the toxic effects of various imidazolium-based ionic liquids (ILs) with varying hydrocarbon chain lengths, on different human cell lines. Multiple biological assays have shown that the ILs with long hydrocarbon chains have stronger adverse effect especially on human liver cancer cells (Huh-7.5 cells). Further, our study has confirmed that the ILs induce necrosis dependent cell death and that it is related to cell membrane damage. To understand the molecular mechanism of such an effect, the cellular membranes were mimicked as lipid monolayers formed at the air-water interface and then as lipid bilayer vesicles. The pressure area-isotherms measured from the monolayer have shown that the interaction of ILs with the lipid layer is energetically favourable. The addition of these ILs reduces the in-plane elasticity of the self-assembled molecular layer. Quasielastic neutron scattering data clearly indicate that ILs in liver lipid vesicles significantly affects the dynamics of the lipid, in particular, the lateral motion of the lipids. It has been concluded that the mammalian cell death induced by these ILs is due to the modulated structure and altered physical properties of the cellular membrane.
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Membrana Celular/efeitos dos fármacos , Imidazóis/química , Líquidos Iônicos/farmacologia , Lipídeos de Membrana/química , Morte Celular/efeitos dos fármacos , Membrana Celular/química , Difusão , Elasticidade , Células HCT116 , Células Hep G2 , Humanos , Líquidos Iônicos/química , Líquidos Iônicos/toxicidade , Células MCF-7RESUMO
The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperatures when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation in the lipids, so that the membranes return to a more fluidic state. To probe how introduction of unsaturation leads to reduced membrane fluidity, a model cationic lipid dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behavior of DODAB have been monitored by quasielastic neutron scattering and time-resolved fluorescence measurements. In the coagel phase, fluidity of the lipid bilayer increases significantly in the presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330 K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in the mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperatures (below the phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.
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Cátions/química , Glicerídeos/química , Bicamadas Lipídicas/química , Varredura Diferencial de Calorimetria , Compostos de Amônio Quaternário/químicaRESUMO
A process in which a disordered system of pre-existing molecules generates an organized structure through specific, local interactions among the molecules themselves is termed molecular self-assembly. Micelles, microemulsions, and vesicles are examples of such self-assembled systems where amphiphilic molecules are involved. As the functional properties of these systems (such as wetting and emulsification, release of solubilized drugs, etc.) are dictated by the dynamic behavior of the surfactants at the molecular level, it is of immense interest to investigate these systems for the same. The dynamics in soft matter systems is quite complex, involving different time and length scales. We used a combination of neutron scattering and molecular dynamics simulation studies in probing the dynamic landscape in various self-assembled surfactant aggregates. Neutron scattering experiments were carried out using several spectrometers covering a wide dynamic range to probe motions on different time scales. The interaction between the surfactants can be varied by changing the molecular architecture, counterion concentration, temperature, and so forth. It is important to study the effect of these parameters on the dynamics of surfactants in these aggregates. We have carried out experiments on various ionic (anionic as well as cationic) micelles with varied counterion concentrations, vesicles, and lipid bilayers to unravel the complex dynamic features present in these systems. In this feature article, we will discuss some important results of our recent work on dynamics in these self-assembled surfactant aggregates.
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Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host-betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that ßC1 protein localized to chloroplasts of host cells, and RaLCB lacking ßC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed ßC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors' knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants.
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Begomovirus/fisiologia , Cloroplastos/virologia , Nicotiana/virologia , Fotossíntese , Doenças das Plantas/virologia , Cloroplastos/metabolismo , Nicotiana/metabolismoRESUMO
Viruses are one of the most devastating plant pathogens causing severe economic losses worldwide. RNA silencing is a robust technology to knock down the expression of specific genes. This mechanism can be exploited to generate virus resistant plants through expression of the viral derived sequences. Viruses in turn have evolved to encode suppressors of RNA silencing to combat host defense. Mixed infection of plants is of common occurrence in nature and simultaneous targeting of suppressor(s) of multiple viruses offers an effective strategy. In this study, we have in silico designed siRNAs against suppressors of the two most devastating viruses of tomato, leaf curl causing tomato begomoviruses and Cucumber mosaic virus. Three different siRNA prediction programs were used to evaluate siRNAs generating capability of each sequence and common putative candidate siRNAs were selected fulfilling the stringent parameters. Our results indicated that in the case of each suppressor a particular region of 100-150 base pairs could be source of potent siRNAs referred as hotspots. Expression of these viral hot spots as a single construct in the plants would facilitate development of transgenic plants with a high degree of broad spectrum resistance against multiple viruses.
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Replication initiator protein (Rep) is indispensable for rolling-circle replication of geminiviruses, a group of plant-infecting circular ssDNA viruses. However, the mechanism of DNA unwinding by circular ssDNA virus-encoded helicases is unknown. To understand geminivirus Rep function, we compared the sequence and secondary structure of Rep with those of bovine papillomavirus E1 and employed charged residue-to-alanine scanning mutagenesis to generate a set of single-substitution mutants in Walker A (K227), in Walker B (D261, 262), and within or adjacent to the B' motif (K272, K286 and K289). All mutants were asymptomatic and viral accumulation could not be detected by Southern blotting in both tomato and N. benthamiana plants. Furthermore, the K272 and K289 mutants were deficient in DNA binding and unwinding. Biochemical studies and modelling data based on comparisons with the known structures of SF3 helicases suggest that the conserved lysine (K289) located in a predicted ß-hairpin loop may interact with ssDNA, while lysine 272 in the B' motif (K272) located on the outer surface of the protein is presumably involved in coupling ATP-induced conformational changes to DNA binding. To the best of our knowledge, this is the first time that the roles of the B' motif and the adjacent ß-hairpin loop in geminivirus replication have been elucidated.